Throwaway type chemical sensor

ABSTRACT

An enzyme electrode for a chemical sensor includes an electrode including a pair of working electrodes, a reference electrode and a counter-electrode. A first film is formed on one of the working electrodes, which includes a polyvinyl alcohol and a surface-active agent. A second film is formed on the other working electrode including polyvinyl alcohol, surface-active agent and enzyme. An overcoat film of high polymer electrolyer including a pH buffer is formed on the first and second films. The polymerization degree of the polyvinyl alcohol is from 300 to 3000 and the high polymer electrolyer may be alginic acid, polystyrene sulfonic acid or polyacrylic acid, and the pH buffer is a positive ion having a valence of 1.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.08/284,116 filed Aug. 2, 1994 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an enzyme electrode for a throwawaytype chemical sensor.

2. Description of the Related Art

A sensor which converts a given chemical substance contained in a testspecimen into an electrical signal for measuring a concentration of thechemical substance is known. This type of the sensor is called achemical sensor. For the purposes of easy handling and improving themeasuring accuracy, improved chemical sensors have been proposed. Someof the chemical sensors of this type have reached the stage of practicaluse.

One of the known chemical sensors is a blood-sugar sensor for measuringa value of blood sugar in the blood as a test specimen. This blood-sugarsensor uses an enzyme electrode including a hydrogen peroxide electrodeand an enzyme electrode using an oxidation reduction enzyme forconverting oxygen into hydrogen peroxide. The blood as a test specimenis dropped on the chemical sensor electrically coupled with a measuringinstrument, and a blood sugar value of the blood is measured. After themeasurement, the sensor (electrode) is disconnected from the measuringinstrument to throw away. This chemical sensor of the throwaway type isfree from troublesome work after measurement, such as calibration of thecalibration curve, washing of the electrodes, and the like. In otherwords, the chemical sensor can be handled in a maintenance free manner.Thus, the handling of the chemical sensor is remarkably improved.Further, this throwaway chemical sensor does not have the problem of themeasuring accuracy deterioration according to an insufficient washing orthe like.

FIG. 14 is a perspective view showing a sensor holder with a throwawaychemical sensor set thereto, which was proposed in Japanese UtilityModel Application No. 1-141108 filed by the inventors of the presentPatent Application. As shown in the drawing, the throwaway chemicalsensor is a sensor body 50 containing a plural number (e.g., 10) ofsensor elements S serially arrayed thereon. This sensor strip 50 ishoused in a sensor holder 51. The foremost sensor element of thesechained sensor elements of the sensor body 50 is exposed at the tip 51aof the sensor holder 51. The exposed sensor element is electricallyconnected to a measuring instrument (not shown) through a connector 52and a cord 53. A test specimen is dropped on the exposed sensor elementto measure the concentration of a given chemical substance thereof.After the measurement, a slider 54 of the sensor holder 51 is extendedto project the used sensor element from the tip 51a of the holder sothat the sensor element is cut out and thrown away. When the used sensoris cut out, a new sensor has already been placed at the tip 51a of theholder and ready for the next measurement. In this way, the process ofcutting out and discarding the used or old sensor and anothermeasurement using a new sensor are repeated for successive measurements.

It is confirmed that the throwaway chemical sensor succeeds in improvingthe efficiency and the accuracy of the measurement. However, thethrowaway chemical sensor has still the following problems which have tobe solved.

It is desirable that the sensor body mounted in the holder have a lot ofsensors so as to improve a. In the case of the sensor body illustratedin the drawing, an increase in the number of the sensor elements leadsto an elongation of the sensor collected body. The length of the sensorcollected body that is acceptable for the sensor holder is limited, sothat the number of the sensor elements contained in the sensor collectedbody is also limited. Generally, the calibration value for the sensor isset up for each manufacturing lot, and input to the measuringinstrument. Where the exchange of the sensor collected body with a newone is frequent, it is highly probable that an operator mistakenly usesthe sensor collected body of another lot, and that calibration valuesare mistakenly input to the instrument.

Further, the sensor element is bent and cut out every after measurement.During this cut-out work, the test specimen may accidentally be attachedto portions other than the sensor element. A danger of contamination andinfection by the specimen inevitably exists.

General chemical sensors easily lose their function by moisture. Forthis reason, the chemical sensors must be moisture proof, particularlywhen they have not been used. If the holder 51 is designed so as to havea completely sealed structure, it is very difficult to store the sensorcollected body after unpacking for a long time while maintaining goodperformances.

In addition, enzyme electrodes of the current-detect type which detectsa concentration of a glucose (dextrose) contained in blood or urine areknown. Some of the enzyme electrodes are of an throwaway type. Anexample of the enzyme electrode of this type is disclosed in UnexaminedJapanese Patent Publication No. Hei. 2-245650. The enzyme electrode hassuch a structure that an electrode portion is formed on an insulatingsubstrate, and an enzyme reaction layer is formed on the electrode. Theenzyme reaction layer contains hydrophilic high polymer substance,oxidation reduction enzyme, and electron acceptor.

In the enzyme electrode thus structured, when a test specimen solutionis dropped on the enzyme reaction layer, the oxidation reduction enzymeand the acceptor are dissolved into the test specimen solution so thatthe enzyme reacts with the substrate (glucose) in the specimen solutionto deoxidize the receptor. The concentration of the substrate in thespecimen solution is calculated using an oxidization current valueobtained after the enzyme reaction completes. However, in the enzymeelectrode thus structured, the oxidation reduction enzyme tends to bondto oxygen. Accordingly, the oxygen dissolved and existing in thespecimen solution (this oxygen will be referred to as a dissolvedoxygen) acts antagonistically, so that the reaction progresses under theinfluence of the oxygen, and an error is caused in the measurement.

Another type of enzyme electrode is disclosed in Unexamined JapanesePatent Publication No. Hei. 2-129541. The enzyme electrode disclosed isof the called hydrogen peroxide type. In this electrode, a substrate(glucose) in a test specimen solution reacts with the dissolved oxygenusing an enzyme as a catalyst to generate hydrogen peroxide. Measured isa current generated when the generated hydrogen peroxide is oxidized atthe electrode. The current value thus measured is used for calculatingthe concentration of the substrate in the test specimen solution.

The enzyme electrode of the hydrogen peroxide type uses the dissolvedoxygen in the test specimen solution. Therefore, it is not necessary touse the electron acceptor, which is indispensable to the enzymeelectrode of the current-detect type. No antagonism between thedissolved oxygen and the acceptor takes place in the test specimensolution, thereby eliminating the measurement error problem caused bythe antagonism. Such an advantageous enzyme electrode of the hydrogenperoxide type has still the following technical problem to be solved,however.

In the case of the enzyme electrode of the hydrogen peroxide type, thesubstrate reacts with the dissolved oxygen in a test specimen solution,using the enzyme as the catalyst. During the reaction process, hydrogenions are generated. Also when the hydrogen peroxide is deoxidized,hydrogen ions are generated. By the generated hydrogen, theconcentration of hydrogen ions is varied in the test specimen solution.When the concentration of hydrogen ions is varied, the reproducibilityof a detected current and a detection sensitivity of the sensor becomeworse in accordance with the pH dependency of the enzyme reaction andthe electrode reaction. An accuracy of detecting the concentration of asubstance under measurement is degraded, and the resultant calibrationcurve has a poor linearity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an enzyme electrode ofthe hydrogen peroxide type which improves a detection accuracy of asubstance under measurement by reducing a variation of the concentrationof hydrogen ions.

An enzyme electrode of a chemical sensor according to the presentinvention includes: an electrode portion including a pair of workingelectrodes and a counter electrode; a first film formed on one of theworking electrodes, which includes polyvinyl alcohol and surface-activeagents; a second film formed on the other working electrode, whichincludes polyvinyl alcohol, surface-active agent and an enzyme; and anovercoat film formed on the first and second film, which comprises highpolymer electrolyer including pH buffer.

In the enzyme electrode of the present invention, diffusion of a testspecimen solution is accelerated by a surface-active agent contained inthe first and second films. A preparatory time before the measurementstarts is reduced. Further, a pH buffer contained in the overcoat filmreduces a variation of the concentration of hydrogen ions in the testspecimen solution.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, advantages and features of the present invention will beunderstood when carefully reading the following detailed description inconnection with the accompanying drawings. In the accompanying drawings:

FIG. 1 is a plan view showing a first embodiment of a sensor bodyaccording to the present invention;

FIG. 2 is a side view showing the sensor body as shown in FIG. 1;

FIG. 3 is an enlarged view of the sensor body showing a sensor portionin FIG. 1 which is a construction of a sensor part of the sensor body;

FIG. 4 is a perspective view showing an upper portion of a sensor holderhousing the sensor body;

FIG. 5 is a perspective view showing the holder when a back coverthereof is opened;

FIG. 6 is a partial cross sectional view showing the holder housing thesensor body set therein;

FIG. 7 is a perspective view showing how a positioning plate engages apositioning member;

FIG. 8 is a plan view showing how a connector of the sensor holder isattached to the sensor body;

FIG. 9 is a cross sectional view taken on line I-I' in FIG. 8;

FIG. 10 is a plan view showing a sensor body according to a secondembodiment of the present invention;

FIG. 11 is a cross sectional view taken on line II-II';

FIG. 12 is a plan view showing another sensor element;

FIG. 13 shows a plan view showing a moisture-proof cap to be attached tothe holder;

FIG. 14 is a perspective view showing a chemical sensor holder when ithouses a serial type throwaway chemical sensor;

FIG. 15 is an exploded view in perspective of a chemical sensor holderaccording to a third embodiment of the present invention;

FIG. 16 is a perspective view showing the sensor holder after assembled;

FIG. 17 is a perspective view showing a sensor feed mechanismincorporated into the sensor holder shown in FIG. 15;

FIG. 18(a) is a plan view showing a sensor body according to a thirdembodiment of the present invention;

FIG. 18(b) is a plan view showing another pattern of a sensor element;

FIG. 19(a) is a plan view showing an enzyme electrode according to anembodiment of the present invention;

FIG. 19(b) is a plan view showing an enzyme electrode collected bodycontaining a plural number of the enzyme electrodes of FIG. 19(a), whichare serially arrayed on the enzyme electrode collected body;

FIG. 20 is a cross sectional view taken on line III--III in FIG. 19(b);

FIG. 21 is a graph showing a calibration curve of the enzyme electrodeof the invention;

FIG. 22 is a graph explaining a first method of applying voltage to theenzyme electrode according to the present invention;

FIG. 23 is a graph explaining a second method of applying voltage to theenzyme electrode according to the present invention;

FIG. 24 is a graph useful in explaining a third method of applyingvoltage to the enzyme electrode according to the present invention; and

FIG. 25 is a graph showing the results of an experiment for confirmingthe useful effects attained by the third voltage applying method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred embodiments of the present invention will be describedwith reference to the accompanying drawings as follows. In thespecification, words indicating directions, locations, and the like,such as right, left, upper and lower, are used to indicate those whenviewed in the drawings. Further, throughout the drawings, like orequivalent portions are designated by like reference numerals.

A structure of a sensor is illustrated in FIGS. 1 to 3. As shown in FIG.1, a disk-shaped sensor body 1 contains a plural number of elementalsensors 2 (referred to as sensor elements) radially extended outwardfrom the circumference thereof. The sensor body 1 is made of a suitableinsulating material, which is conventionally used for various types ofsubstrate. The insulating material may be a plastic as epoxy resin orglass epoxy resin, or an equivalent material. The circumferentialportion of the sensor body 1 is shaped to have V-shaped notches 3equidistantly and angularly arrayed therearound. Of these notches 3, theadjacent notches define a trapezoidal part. Thus, the notches 3 andtrapezoidal parts are alternately arrayed on the circumference of thesensor body 1. The sensor elements 2 are formed in the trapezoidalparts, respectively.

A pattern of sensor elements 2 formed in the trapezoidal part isillustrated in detail in FIG. 3. Each of the sensor elements 2 is formedby forming a circuit pattern of conductive material on the substrate, orthe trapezoidal part, as with conventional sensors. A preferableconductive material may be platinum (Pt). A conventional, suitableplating technique, such as a platinum plating process or conductivematerial printing process, may be used for forming the sensor circuit.

In the sensor circuit, a sensor portion is formed of a counter electrode6, a reference electrode 7, a first working electrode 8, and a secondworking electrode 9. Reference numerals 10a, 10b, 10c, and 10d designateterminals of those electrodes. One sensor element 2 is formed by thesensor portions and the terminal portions. The sensor body 1 is shapedlike a disk with the fringe made of a number of the trapezoidal partseach having one sensor element 2 formed therein.

Reference numeral 4 designates a positioning plate coaxial with thesensor body 1. The positioning plate 4 is also used for holding adesiccant. Accordingly, the positioning plate 4 needs to be made ofair-permeable material, such as a plastic material with a number ofperforations or an air-permeable nonplastic material, e.g., hard unwovenfabric. The material has to be strong enough to withstand the operationof the positioning plate to be given later. The positioning plate 4 thusconstructed is filled with desiccant. Grooves 4a are equidistantlyformed in the circumferential outer face, so that the positioning plate4 takes the form of a gear. The positioning plate 4 may consist of twohalf-circular plates, which are combined and fixed to thepositioning-plate location on the sensor body 1. The positioning plate 4may also be formed by fitting a positioning plate into an openingpreviously formed in the sensor body 1. A knob 5, which is coaxial withthe sensor body 1, is provided to the surface of the sensor body 1having the sensor element 2 formed thereon.

FIGS. 4 and 5 shows the structure of a holder for holding the sensorbody 1. FIG. 1 is a perspective view showing the upper part of theholder when it holds the sensor body 1 therein, and FIG. 2 is aperspective view showing the holder when a back cover thereof is opened,the holder being illustrated upside down.

Referring mainly to FIG. 5, a sensor holder 11 has a space where thesensor body 1 is disposed. A sensor support portion (hereinafter,referred to as a first support) 13, shaped like a ring, has an opening13a at the central part thereof. The opening 13a is for inserting theknob. The surface of the first support 13 is formed to have a smallslide resistance.

A positioning member 14 is located adjacent the first support 13. Thepositioning member 14 includes a support 14a (referring to FIG. 7) and astopper 14b attached to the support 14a. The support 14a is erected onthe rear side of the top surface 11a of the holder 11 (in FIG. 5, theholder is illustrated with a back cover 12 thereof undermost.). Thestopper 14b is constantly urged outward by a coiled spring (not shown)contained therein. The stopper 14b, when receiving a pushing force, isput into the support 14a while resisting the resilient force of thecoiled spring.

The back cover 12 is provided with a second support 15 as a secondmember for supporting the sensor body 1. The second support 15 includesa flat ring-shaped member 15a and a pushing member 15b disposed withinthe ring-shaped member 15a so that it is projected from the member 15aby a coiled spring 16 (FIG. 6). When the back cover 12 is closed, thesecond support 15 becomes coaxial with the first support 13.

As illustrated in FIGS. 4 and 5, the sensor holder 11 becomes thintoward the fore end thereof (left side in the drawing). An opening 18 ofa V-shaped (as viewed from top) cutout is formed at the fore end of thesensor holder 11. The opening 18 allows only one of the sensor elements2 of the sensor body 1 contained to the holder 11 to be exposed in theoutside of the holder.

Reference numeral 17 designates a connector provided on the rear side ofthe top surface 11a of the holder 11. The connector 17 includesconnector terminals 17a to be respectively connected to the terminals10a to 10d of each sensor element 2, and a terminal holder 17b forholding these connector terminals (also see FIGS. 8 and 9). Cords 19 areconnected at the first ends to the connector 17 while at the second endsto a measuring instrument which is not shown.

The operation and the handling of the throwaway sensor thus constructedwill be described as follows.

The sensor body 1 which is not used is packed in a moisture-proof state.If the packed sensor body 1 is well sealed, the sensor body 1 can besufficiently protected from moisture without using desiccant, whichotherwise would be put into the positioning plate 4 of the sensor body1.

At the beginning of use of the sensor body, the desiccant is removedfrom the sensor body 1. On the other hand, the back cover 12 of thesensor holder 11 is opened as shown in FIG. 5. The sensor body 1 is setto the holder 11 such that the sensor-element formed surface thereof isfaced down (in FIG. 5), that is, the sensor elements 2 face the topsurface 11a of the holder 11, and the knob 5 of the sensor body 1 ispassed through the opening 13a of the first support 13 till it isprotrude from the holder. In this state, the stopper 14b of thepositioning member 14 engages with one of the grooves 4a of thepositioning plate 4.

After the sensor body 1 is thus set in the holder, the back cover 12 isclosed. In this state, as shown in FIG. 6, the pushing member 15b of thesecond support 15 provided on the back cover 12 is pressed against thesurface of the positioning plate 4 of the sensor body 1, and cooperateswith the first support to rotatably support the sensor body 1 at a givenlocation in the sensor holder 11.

The sensor body 1 is disposed in the holder 11 such that one of theplural number of sensor elements 2 forming the sensor body 1 is partlyexposed to the outside of the holder 11 (FIG. 4). More specifically, thesensor part of the sensor element, which includes the electrodes 6, 7,8, and 9, is exposed to the outside through the V-shaped opening 18 ofthe sensor holder 11. The terminals 10a to 10d of the sensor element 2the sensor part of which is exposed outside are brought into contactwith the connector terminals 17a of the connector 17, respectively. As aresult, this sensor element 2 is electrically connected to the measuringinstrument, through the connector 17 and the cords 19. The holder 11containing the sensor body 1 thus set therein is placed in a so that theterminals of the sensor element 2 are located on the obverse side of theholder, as shown in FIG. 4. Then, for measurement, a test piece isattached to the sensor part of the sensor element 2.

After the measurement is completed, the knob 5 is turned. With the turnof the knob, the stopper 14b of the positioning member 14 is pushed intothe support 14a thereof while resisting the resilient force of thespring contained therein. When the next groove 4a reaches in front ofthe support 14a, the support 14a is pushed by the spring in the reversedirection and put into the groove 4a. Then, a new sensor element 2 ofthe sensor body 1, located adjacent the already used sensor element 2,is set at the opening 18 and comes in contact with the connector 17 asfor the previous sensor. In this way, new sensor elements 2 aresuccessively set at the opening for the successive measurements.

When the measurement progresses in this way and all of the sensorelements 2 of the sensor body 1 are used, an operator opens the backcover 12 of the sensor holder 11 so as to replace the old sensor body 1with a new one. When the measurement is completed and the sensor body 1still contains the sensor elements 2 not yet used, the holder 11 ispacked and stored in a moisture-proof state for another measurement.

In the case where the measurement is completed and the sensor body 1still includes new sensor elements 2, should not be taken the sensorbody 1 out of the holder 11. When it is taken out of the holder, the notyet used sensor elements 2 are touched with finger tips so as to reducethe reliability of the sensor elements 2. Accordingly, it is desirableto store a sensor body 1 having not yet used sensor elements 2 whileleaving the sensor holder 11 therein. For the case where storing thesensor holder 11, the positioning plate 4 of the sensor body 1preferably contains desiccant so that the inside of the holder 11 iskept in a satisfactorily dried state.

A second embodiment of a throwaway sensor according to the presentinvention will be described with reference to FIGS. 10 and 11.

While the first embodiment uses the positioning plate 4 for positioningthe sensor part, the second embodiment uses a positioning device inwhich the fringe of the sensor body 1 is configured to have the V-shapednotches 3 and the trapezoidal parts on which the sensor elements 2 areto be formed. A sensor body 1, shaped like a ring as viewed in plan,includes an opening 1a formed at the central part and key grooves 1bformed in the side wall.

The sensor holder 11 is provided with a device for rotatably supportingthe sensor body 1. A main shaft 31 is rotatably planted on a bottomplate 11b of the holder 11. The top end of the main shaft 31 serves as aknob for turning the sensor body, which corresponds to the knob 5 in thefirst embodiment. An engaging shaft 32 is attached to the main shaft 31.The diameter of the engaging shaft 32 is substantially equal to theinner diameter of the opening 1a of the sensor body 1. A plural numberof keys 32a are protruded from the side wall of the engaging shaft 32 soas to respectively engage the key grooves 1b of the sensor body 1. Asupport plate 33 is coaxial with the main shaft 31 and the engagingshaft 32. The support plate 33 is larger in diameter than the engagingshaft 32. The support plate 33 supports the sensor body 1 which has beenfitted to the engaging shaft 32. The positioning member 14 is mounted ata location adjacent to the circumferential edge of the sensor body 1 onthe bottom plate 11b so that the stopper 14b of the positioning member14 engages any of the notches 3. When the stopper 14b engages one of thenotches 3, a sensor element 2 specified by the notch engaging thestopper is set at the opening 18 of the sensor holder 11. In otherwords, the positioning member 14 positions the sensor body 1.

After the sensor body 1 is set to the holder 11, a cover 30 is closed sothat the main shaft 31 is partly protruded from the cover 30. Aspreviously mentioned, in the first embodiment, the cover is the backcover serving as the bottom plate. In the second embodiment, the bottomplate is integral with the holder 11, and the top surface of the holderis used for the cover 30. A desiccant 34 is located within the sensorholder 11. With use of the desiccant, the sensor body 1 in the holder 11is protected from moisture as in the first embodiment.

Another construction of the sensor elements 2 (FIG. 3) will be describedwith reference to FIG. 12.

In the figure, reference numeral 40 designates a reference electrode;41a, 41b, and 41c, counter electrodes; 42a and 42b, working electrodes;43a, 43b, 43c, and 43d, terminals; and 44 lead wires for connectingthose electrodes. A calibrating part 45 consists of a set of electrodes(in this instance, three electrodes forms one set of the electrodes).The calibrating part 45 is formed on one of the trapezoidal parts of thesensor body 1, and is provided for calibrating the sensor.

Next, FIG. 13 is a plan view showing a moisture-proof cap to be attachedto the chemical sensor holder 11. A moisture-proof cap 20 is made ofhighly water-proof material, such as plastics. The moisture-proof cap 20includes a pair of stopper pawls 20a and 20b formed at the opening forreceiving the holder. A pair of stopper pawls 20a and 20b which isformed at the upper and lower of the holder receiving opening side,respectively, is engaged with a pair of groove 21 formed in the sensorholder 11 so that the moisture-proof cap 20 is airtightly connected withthe holder 11. The moisture-proof cap 20 is very convenient for sealingthe holder containing the sensor body having the not-yet-used sensorelements for long term storage of the sensor. The moisture-proof cap 20may also be made of water-proof and flexible material, such as rubber.

In the embodiments mentioned above, the positioning plate 4 has twofunctions: 1.) a water-proofing function by desiccant and 2.) apositioning function for positioning the sensor body 1. If required, thedesiccant is located at another place in the sensor holder 11, and thepositioning plate 4 is used only for positioning the sensor body 1. Inthis case, the material of the positioning plate 4 is not limited tothat referred to above.

FIGS. 15 to 17 show a third embodiment of a chemical sensor holderaccording to the present invention. FIG. 15 is an exploded view inperspective of a chemical sensor holder according to the thirdembodiment. In a throwaway type chemical sensor 101, a sensor jacket 104includes an upper cover 102 with a dropping part 116 and a lower cover103. A sensor body 105 and a sensor cover 106 bonded to the sensor body105 are sandwiched between the upper and the lower covers 102 and 103. Anumber of sensor elements 144 are radially mounted on the sensor body105 as will be described later. The sensor cover 106 includespositioning grooves 107 formed in the circumferential edge thereof. Whena protrusion 114 of a holder/sensor feed mechanism 118 (FIG. 17), whichwill be described with reference to FIG. 17, engages one of thepositioning grooves 107, the sensor body 105 is turned in a givendirection. The lower cover 103 is made up of a ratchet 109 for turningthe sensor body 105 every groove while preventing reversal of the turn,a pushing portion 110, a hole 108 for receiving the protrusion 114, anda sensor rotation window 111. To assemble the chemical sensor 101, theupper and lower covers 102 and 103 are coupled and fastened bycounter-sunk screws inserted into holes 112 of the upper cover 102 andholes 113 of the lower cover 103. The chemical sensor 101 thus assembledis illustrated in FIG. 16.

The chemical sensor 101 thus assembled is positioned relative to theholder/sensor feed mechanism 118 shown in FIG. 17. The holder/sensorfeed mechanism 118 is provided with a contact portion 117, a sensorlever 115 for moving the sensor, and the protrusion 114. The protrusion114, interlocked with the sensor lever 115, is set at an initialposition by a spring which is not shown. When the sensor lever 115 ispushed, the protrusion 114 is moved in the direction of an arrow, viz.,in the sensor moving direction, while resisting the resilient force ofthe spring. When the sensor lever 115 is released, the protrusion 114 isreturned to the initial position by the resilient force of the spring.The protrusion 114 is obliquely raised to have a slanted surface and avertical surface. When the chemical sensor 101 is set to theholder/sensor feed mechanism 118, the protrusion 114 engages at thevertical surface one of the positioning grooves 107 through the hole108, and turns the disk-shaped sensor cover 106 unidirectionally. Thecontact portion 117 is brought into contact with the terminals of eachsensor element on the sensor body 105 through a contact window 119. Oneof the sensor elements arrayed on the sensor body 105 is located at thedropping part 116 (FIG. 16). A test specimen is dropped onto the exposedsensor element. The result is transferred in the form of an electricalsignal to a measuring instrument which is not shown.

A construction of the sensor body 105 of the throwaway chemical sensoris as shown in FIGS. 18(a) and 18(b). A plural number of sensor elements144 each as shown in FIG. 18(b) are radially formed on a disk-shapedsupport member. As shown in the drawings, each of the sensor elements144 includes a reference electrode 140, its terminal 143b, counterelectrodes 141a, 141b, and 141c, first and second working electrodes142a and 142b, and their terminals 143c and 143d. The functions of theseelectrodes have already been described in the abovementionedembodiments, and hence no further description thereof will be given. Thesensor body 105 has also a calibrating part 145 as in the previousembodiment. The calibrating part 145 also consists of a set ofelectrodes (in this instance, three electrodes forms one set of theelectrodes). The calibrating part 145 is formed on one of thetrapezoidal parts of the sensor body 105, and is provided forcalibrating the sensor.

Since the sensor elements 144 are radially disposed on the disk-likesupport, the number of sensor elements is increased for a fixed size ofthe sensor body. The sensor body does not need to be as frequently setto the holder for its exchange with a new one. This improves theworkability and reliability of the sensor. Additionally, the troublesomework to exchange the sensor element every measurement is eliminated.Since the test specimen will not be attached to portions other than thesensor element when it is exchanged, there is eliminated the danger ofcontamination and infection by the test specimen mistakenly attached.

A specific enzyme electrode as a chemical sensor, which is adaptable forthe sensor holders of the present invention as mentioned above, will bedescribed with reference to FIGS. 19 to 21. A plural number of theenzyme electrodes may be serially arrayed on a strip-shaped sensor body,as shown in FIG. 19(b) or radially arrayed on a disk-shaped sensor bodyas shown in FIG. 1 or 15. As a matter of course, it may be used as asingle enzyme electrode as shown in FIG. 19(a).

Referring to FIG. 19(a), there is shown an enzyme electrode according tothe present invention. In the drawing, the enzyme electrode 201 includesan electrode portion 204 of a predetermined pattern, which is formed onan insulating substrate 203 by etching process, for example. Theelectrode portion 204 of the enzyme electrode 201 includes a referenceelectrode 204a located at the central part on the insulating substrate203, first and second working electrodes 204b and 204c located on bothsides of the reference electrode 204a, and a counter electrode 204dlocated on the right side of the working electrodes 204b and 204c. Thecounter electrode 204d has upper and lower connection wires extendedfrom the top and bottom of the left side thereof. The upper connectionwire is extended above the first working electrode 204b and terminatedbefore the terminals 205. The lower connection wire is extended belowthe second working electrode 204c and continuous to the related terminal205.

These electrodes 204a to 204d are respectively connected to terminals205 located on the left end portion. Terminals 205 are connected to acontact terminal of a measuring instrument, not shown, when a testspecimen is subjected to a measurement. For the measurement, the testspecimen is dropped on the electrode portion. An insulating film 206having a substantially U-shape when viewed from top, covers an areaincluding a part of the counter electrode 204d and a portion forconnecting the electrodes 204a to 204d to the terminals 205.

A first film 207 is layered on the first working electrode 204b. Asecond film 208 is layered on the second working electrode 204c. Anovercoat film 209 is further layered on both the first and second films207 and 208. The first film 207 contains at least polyvinyl alcohol andsurface-active agent. An example of the composition of the first film207 is given below. In the composition, the components are expressed byweight for the unit area of 1 mm² of the film.

EXAMPLE

    ______________________________________                                        (Composition of the first film 207)                                           ______________________________________                                        1)  Polyvinyl alcohol of 300 to 3000 in                                                                    0.3 μg to 3.0 μg                               polymerization degree                                                     2)  SDS (surface-active agent)                                                                             0.5 μg to 1.5 μg                           3)  Sodium alginate          0.12 μg to 0.4 μg                          4)  Phosphoric acid buffer                                                        *Dipotassium hydrogen phosphate                                                                        0 μg to 11.8 μg                                *Sodium hydrogen phosphate                                                                             0 μg to 4.5 μg                             ______________________________________                                    

The reason why the polymerization degree of the polyvinyl alcohol isselected in the range of 300 to 3000 is as follows. Polyvinyl alcohol ishard to dissolve in water, if its polymerization degree is high.Surface-active agent (SDS: dodecyl sodium sulfate) and phosphoric acidbuffer are nonuniformly mixed so that the polyvinyl alcohol is separatedout of the solution. Particularly, when the polymerization degreeexceeds 3000, these components are separated out of a solution suitablefor film formation even at the lower limit value (0.3 μg) of thecomposition example. The film components are nonuniformly distributed tocause an error in measurement. If its polymerization degree is low, thesolubility of polyvinyl alcohol is increased so that it fails to graspthe enzyme within a measuring time. As a result, the measurementreproducibility is deteriorated.

Ideally, it is desirable to keep a high buffering action also in theenzyme fixing film (polyvinyl alcohol film). However, polyvinyl alcoholis separated out as referred to above. To avoid the separation ofpolyvinyl alcohol, there is a limit in selecting the polymerizationdegree of polyvinyl alcohol. When the polymerization degree of thepolyvinyl alcohol is 300, SDS, sodium alginate, dipotassium hydrogenphosphate, and sodium hydrogen phosphate may be contained up to theupper limit values (3.0 μg).

When the polymerization degree of polyvinyl alcohol is 3000, thesecomponents may be contained only up to the lower limit value (0.3 μg).In consideration of the polymerization degree in connection with themeasuring time, for a long measuring time, the polymerization degree isdesirably high, while for a short measuring time, it is desirably low.The reason why the content of the polyvinyl alcohol is selected to bebetween 0.3 μg and 3.0 μg will be described. The polyvinyl alcohol ofhigh polymerization degree (3000), even if its amount is small, has astrength high enough to satisfactorily hold enzyme during the measuringtime. If its amount is too small, however, it cannot grasp a necessaryamount of enzyme, so that the enzyme is eluted. Accordingly, the minimumcontent of the polyvinyl alcohol is 0.3 μg.

When the polymerization degree of the polyvinyl alcohol is low (300),the absorption rate of the polyvinyl alcohol is higher than that when itis high. Therefore, a large amount of enzyme can be grasped byincreasing the thickness of the first film. If the film is too thick,its response speed varies, leading to deterioration of the measuringaccuracy. This unwanted variation of the response speed becomesremarkable when its content is in excess of 3.0 μg. To avoid this, thecontent of the polyvinyl alcohol is set to below 3.0 μg.

The second film 208 contains at least polyvinyl alcohol, surface-activeagent, and enzyme. An example of the composition of the film containsglucose oxidase of 0.5 units/mm² in addition to the composition of thefirst film 207.

The overcoat film 209 contains at least high polymer electrolytecontaining pH buffer. An example of the composition of the overcoat filmis shown below.

EXAMPLE

    ______________________________________                                        (Composition of the overcoat film 209)                                        ______________________________________                                        1)  Sodium alginate (high polymer electrolyte)                                                              5 μg to 20 μg                             2)  Phosphoric acid buffer composition by molar ratio                             *Dipotassium hydrogen phosphate:Sodium hydrogen                               phosphate (sodium phosphate) = 1:1 to 9:1                                     *Dipotassium hydrogen phosphate                                                                         32 μg to 236 μg                               *Sodium hydrogen phosphate                                                                              4.5 μg to 90 μg                           ______________________________________                                    

The high polymer electrolyte of the overcoat film 209 may be any othersuitable material than alginic acid, for example, polystyrene sulfonicacid or polyacrylic acid. The surface-active agent used for the firstand second films 207 and 208 may be any other suitable material thanSDS, e.g., any negative ion active agent, such as higher fatty acidalkaline salts and alkyl aryl sulfonic acid salts, or any nonionicsurface-active agent, such as polyethylene glycol alkyl phenyl ether andsorbitan fatty acid ester.

The pH buffer may be not only the phosphoric acid but also a reagentsatisfying the following conditions. Positive ions having a valence of 2or more are not contained. When it is dissolved into a test specimensolution, the concentration of hydrogen ions is between 5 and 8. Thereagent does not hinder the enzyme reaction and the electrode reaction.Use of a pH buffer not containing the positive ions having a valence of2 or more is preferable since the pH buffer containing such positiveions makes the coating work of gelatinized alginic acid difficult.

A further description of the overcoat film 209 will be given. Thequantity of the sodium alginate is determined by the thickness of thefilm and a mixing ratio of the sodium alginate and the buffer at which auniform distribution of the buffer is secured. If the buffer isnonuniformly distributed in the sodium alginate, much time is taken fordissolving the buffer into the sodium alginate so that the concentrationdistribution varies. The variation of the concentration distributionadversely affects the measuring accuracy. A high buffering action isdesirable; however, it must be properly selected allowing for themeasuring accuracy and the measuring time. The quantity of the sodiumalginate determines the thickness of the overcoat film. If the film istoo thick, time taken for the film to absorb the test specimen solutionis long. Conversely, if it is too thin, the separation ability ofprotein substance and blood corpuscles from the test specimen solutionbecomes lower so that the measuring accuracy is also worsened.Generally, this type of the electrode is designed such that theabsorbing time of the test specimen solution is within one minute.

In the example of the composition of the overcoat film 209, dipotassiumhydrogen phosphate and sodium hydrogen phosphate are used, because thepH value, as desired, can be changed by adjusting the composition ratioof these materials. When these materials are mixed, at 1:1, in thesodium alginate, the pH value which indicates a buffering action can beadjusted to be 5.2. When these materials are mixed at 9:1, the adjustedpH value is approximately 7.8. Thus, by properly selecting the quantityof the buffer and the composition ratio of the buffering materials, anactivity of the enzyme is controlled, whereby a calibration curve can bechanged.

When the composition ratio is 1:1 and the quantity of the buffer is low,the sensitivity of the sensor is increased at a low substrateconcentration, but it is decreased at a high substrate concentration.When the composition ratio is 9:1 and the quantity of the buffer ishigh, the sensitivity is decreased at a low substrate concentration, butit is increased at a high substrate concentration.

A specific example of an enzyme electrode according to the presentinvention will be described.

An enzyme electrode structured like the enzyme electrode 201 shown inFIG. 1 was manufactured. First and second films 207 and 208, and anovercoat film 209 were fabricated at the following composition ratios.Those films fabricated were layered on an electrode portion 204. In thefollowing composition ratios, the respective components are expressed atthe ratios in distilled water of 1 ml.

    ______________________________________                                        A. Components of the first film 207                                           ______________________________________                                        1)   Polyvinyl alcohol of 500 in polymerization degree                                                       2.8 mg/ml                                      2)   SDS (surface-active agent)                                                                              2.5 mg/ml                                      3)   Sodium alginate           0.5 mg/ml                                      4)   Phosphoric acid buffer     30 mM                                              *Dipotassium hydrogen phosphate                                                                         3.5 mg/ml                                           *Sodium hydrogen phosphate                                                                              1.2 mg/ml                                      ______________________________________                                    

B. Components of the second film 208

The second film 208 includes glucose oxidase of 500 units/ml in additionto the components of the first film 207.

    ______________________________________                                        C. Components of the overcoat film 209                                        ______________________________________                                        1)   Sodium alginate (high polymer electrolyte)                                                               10 mg/ml                                      2)   Phosphoric acid buffer     0.6 M                                              Phosphoric acid buffer composition by molar ratio                             *Dipotassium hydrogen phosphate:Sodium hydrogen                               phosphate (sodium phosphate) = 2:1                                            *Dipotassium hydrogen phosphate                                                                         116 mg/ml                                           *Sodium hydrogen phosphate                                                                               40 mg/ml                                      ______________________________________                                    

The first working electrode 204b was coated with the thus prepared filmmaterial of the first film 207 of 2 μl, by a dispenser. Thereafter, itwas placed in a desiccator to be dried for 20 minutes. The secondworking electrode 204c was coated with the same amount of the thusprepared film material of the second film 208 to be dried in the sameconditions. Thereafter, the first and second films 207 and 208 werecoated with the thus prepared film material of the overcoat film 209 of8 μl, and dried for one hour or longer.

An occult blood as a test specimen solution was dropped on the enzymeelectrode thus manufactured. The resultant calibration curve was asshown in FIG. 21. As seen from the drawing, the calibration curve of theenzyme electrode of the invention has an excellent linearity.

From the foregoing description, in the enzyme electrode of theinvention, the first film formed on the first working electrode containssurface-active agent. With the use of the surface-active agent,diffusion of the test specimen solution is accelerated by thesurface-active agent. A preparatory time before a measurement starts isreduced. Further, since the overcoat film contains pH buffer, if thesubstrate in the test specimen solution reacts with the dissolved oxygento generate hydrogen, a variation of the concentration of hydrogen ionsis reduced by the pH buffer, thereby providing a high accuracy ofmeasurement.

Further, in the enzyme electrode of the hydrogen peroxide type, whichincludes the enzyme electrode of the invention, a range allowing asubstrate to be measured is limited by the dissolved oxygen in the testspecimen solution. To expand this range, the inventor developed someinventive and unique methods of applying voltage to the enzymeelectrode, which can expand this range. These methods applied to theelectrode will be described with reference to FIGS. 22 to 25.

FIG. 22 is a graph showing a first method of applying voltage to theenzyme electrode. The voltage applying method includes first to fourthsteps. The first step is for detecting the contact of a test specimenwith the enzyme electrode 201. To detect this contact, a positivepotential V₁ is applied to the first and second working electrodes 204band 204c. Current I₁ flowing a path between the first and second workingelectrodes 204b and 204c and the counter electrode 204d is detected.

The potential V₁ applied to the first and second working electrodes 204band 204c may be negative in polarity. The amplitude of the potential V₁is preferably as low as possible in order to minimize an adverseinfluence on the electrodes and the enzyme films. The polarity of thecurrent I₁ depends on a state of the electrode and the direction ofimpregnating the test specimen into the electrodes. Therefore, it isdesirable to use a current detecting device capable of detectingpositive and negative currents.

A second step, following the first step, keeps the potential applied tothe sensor at a first potential for a preset time t₁ at which no currentflows into the first and second working electrodes 204b and 204c. Inusing the enzyme electrode 201 as the throwaway chemical sensor, afterthe test specimen is dropped on the electrode portion 204, it isnecessary to sufficiently adapt the dropped test specimen to the enzymefilms and others such as reagent in an enzyme reaction area on thesurface of the electrode portion 204. Accordingly, a time zone isprovided where no current flows into the electrode portion 204 for thepreset time t₁ after the test specimen is detected.

The first potential for causing no current to flow into the electrodeportion 204 may be realized by setting the potential applied to theelectrode portion 204 at substantially 0, or disconnecting a potentialapplying device (power source) from the electrode portion 204. At thistime, the same may also be realized by using such a voltage as to causean extremely smaller current than the measurement detection current. Thepreset time t₁, usually 15 to 40 seconds, preferably 30 to 40 second, isdetermined by the composition and the thickness of the enzyme film, thestructure of the electrode, and the like. This time may be reduced bymaking the film thickness as thin as possible, constructing theelectrode structure so as to swiftly guide the test specimen onto thesurface of the enzyme film, using such a material as to well absorb thetest specimen, or the like.

A third step, following the second step, is to apply a second potentialV₂, which is higher than a hydrogen peroxide detect potential (indicatedby a phantom line in FIG. 22), to the first and second workingelectrodes 204b and 204c for another preset time t₂, and to drop thepotential to a third potential V₃ below zero potential. In thisinstance, the hydrogen peroxide detect potential is approximately 600mV, although it depends on the structure of the electrode portion 204.

The time t₂ for applying the second potential V₂ is preferably as shortas possible in order to remove adverse effects on the reproducibility ofthe measuring current, although it depends on the amplitude of thesecond potential V₂. The third potential V₃ may be any potential if itis below zero potential; however, if the hydrogen peroxide detectpotential is 600 mV, the third potential is preferably set to apotential 800 to 100 mV lower than the hydrogen peroxide detectpotential.

In a fourth step following the third step, the potential applied to thefirst and second working electrodes 204b and 204c is swept up from thethird potential V₃ to a fourth potential V_(end) higher than thehydrogen peroxide detect potential, at a fixed rate Vs. The sweepingrate Vs may be set at a proper value, preferably substantially 100mV/sec. A peak value of current in the range of the voltage sweep up tothe fourth potential Vend, a current value at a potential separated fromthe potential causing the peak current value by a preset potentialvalue, or a current value at a specific potential is detected, and thedetected current value is converted into a concentration of a substanceunder measurement by using a calibration curve previously charted.

The voltage applying method produces the results comparable with thosewhen the dissolved oxygen is increased, as will be seen from theexperiment results. Although the mechanism causing such results cannotbe clearly explained at present stage, it is thought that theelectrolysis progresses under the applied voltage to supply enzyme andhence to expand a range allowing the concentration of the substanceunder measurement to be measured.

FIG. 23 is a graph explaining a second method applying voltage to theenzyme electrode according to the present invention. Only the differentportions of the second voltage applying method from the first voltageapplying method will be described. The second voltage applying methodincludes first to fourth steps as the first voltage applying method. Ofthose steps, the first, second, and fourth steps are the same as thoseof the first voltage applying method.

In the third step, a potential causing a large fixed current I₂ isapplied to the first and second working electrodes 204b and 204c for apreset time t₂, and then the potential is decreased to the thirdpotential V₃ below zero potential. The fixed current I₂ is larger thanan expected peak hydrogen peroxide detect current. Where the measurablemaximum concentration of glucose is 500 mg/dl, the current correspondingto the concentration is the expected peak hydrogen peroxide detectcurrent (in this instance, it is set at approximately 40 μA). In thisstep, the current control is carried out. Accordingly, the secondvoltage applying method is suitable for the chemical sensor in which thereference electrode 204a and the first and second working electrodes204b and 204c are made of the same material. The reference potential atthe reference electrode 204a depends on the components of the testspecimen, and indicates a relative potential.

In this case, if the equal potential is applied to the sensor pluraltimes, the same electrode reaction does not always recur. In ameasurement, the hydrogen peroxide detect voltage is 600 mV and inanother measurement it may be 800 mV. The voltage applying method inwhich a fixed voltage is applied to the sensor is unsatisfactory inproviding a good measurement reproducibility. To cope with this, thesecond voltage applying method uses the step to flow a fixed current I₂into the sensor for a fixed time t₂. With this step, a quantity ofchemical reaction at the electrodes is controlled to be constant so thatthe measurement reproducibility is improved. The fixed time t₂ is setunder the conditions similar to those in the first voltage applyingmethod.

FIG. 24 is a graph explaining a third method of applying voltage to theenzyme electrode according to the present invention. Only the differentportions of the third voltage applying method from the first and secondvoltage applying methods will be described. The third voltage applyingmethod includes first to fourth steps as the first and second voltageapplying methods. Of those steps, the first, second, and fourth stepsare the same as those of the first and second voltage applying methods.

In the third step, a potential causing a current larger than a hydrogenperoxide detect current is applied to the first and second workingelectrodes 204b and 204c for a preset time t₂. Succeedingly, thepotential that is reached when the time duration terminates is kept fora preset time t₃. Finally, the potential is decreased to the thirdpotential V₃ below zero potential. The measuring time of the thirdvoltage applying method is slightly longer than that of the first andsecond ones. However, the third voltage applying method can expand arange allowing the concentration of the substance under measurement tobe measured, and improve the measurement reproducibility.

FIG. 25 is a graph showing the results of an experiment for confirmingthe effects of the voltage applying methods described above. In theexperiment, enzyme electrodes of the composition ratios as mentionedabove were manufactured and tested under the following conditions.

The width w and the length l of each of the first and second workingelectrodes 204b and 204c were: w=0.5 mm and l=2.5 mm. A test specimenwas the blood of a cattle. EDTA2kl of 3 mg/ml was added to the blood toadjust the glucose. Voltage, Current or time which was applied in theexperiment is indicated as follows.

V₁ : 200 mV

I₁ : 5 μA

t₁ : 20 sec

V₂ : 1500 mV

I₂ : 100, 120, 200 μA

t₂ : 5 sec

t₃ : 2 sec

V₃ : -400 mV

Vs: 100 mV/sec

The curves of FIG. 25 were plotted when voltage was applied to thesensor by the third voltage applying method. In the graph, a curve (1)was plotted in a condition that the third step of the third voltageapplying method was omitted, and in the fourth step voltage wasincreased from 0. A curve (2) was plotted when the potential V₃ was setat 0. Curves (3) to (5) were plotted when the current I₂ in the thirdstep was set at 100, 120, and 200 μA.

As seen from FIG. 25, in the curve (1), the glucose concentration issaturated at 180 mg/dl. On the other hand, when the voltage V₃ is set tobelow 0 V and the current I₂ is increased, the saturation value of theglucose concentration is increased, thereby expanding the measurablerange.

According to the foregoing description, the voltage applying methods ofthe present invention can expand the measurable range without making thestructure of the enzyme electrode complicated.

What is claimed is:
 1. An enzyme electrode of a chemical sensorcomprising:an electrode portion including a pair of working electrodes,a reference electrode and a counter electrode; a first film formed onone of said working electrodes, said first film comprising polyvinylalcohol and surface-active agent which accelerates diffusion of a testsample solution through said first film; a second film formed on theother working electrode, said second film comprising polyvinyl alcohol,enzyme and a surface-active agent which accelerates diffusion of a testsample solution through said second film; and an overcoat film formed onsaid first and second film, said overcoat film comprising a high polymerelectrolyte including pH buffer wherein said high polymer electrolyte isselected from the group consisting of polyacrylic acid and water solublepolysaccharide containing carboxyl groups; and wherein said pH bufferreduces variation of the concentration of hydrogen ions in said testsample.
 2. An enzyme electrode of a chemical sensor according to claim1, wherein said polyvinyl alcohol has a degree of polymerization from300 to
 3000. 3. An enzyme electrode of a chemical sensor according toclaim 1, wherein said high polymer electrolyte is selected from thegroup consisting of alginic acid, polystyrene sulfonic acid andpolyacrylic acid.
 4. An enzyme electrode of a chemical sensor accordingto claim 1, wherein said pH buffer includes a positive ion and saidpositive ion has a valence of one.